Method for the production and isolation of 5-phospho-d-ribosyl-1-pyrophosphate, d-ribose-1, 5-diphosphate and d-ribose-5-phosphate



Un t d t s Part? 0.

' METHOD FOR m PRODUCTION AND ISOLA- TION- OF S-PHOSPHO-D-RIBOSYL-l-PYRO- PHOSPHATE, D-RIBOSE 1,5 -DIPHOSPHATE .D-RIBOSE-S-PHOSPHATE v ,Yo'shiaki Kawamori, Masahiko Matsuura,

ABSTRACT OF THE DISCLOSURE The present disclosure is directed to a method of preparing and recovering D-ribose phosphates such as S-phos- 'pho-D-ribosyl-l pyrophosphate (PRPP), D-ribose-l, 5-

diphosphate (PRP) and D-ribose-S-phosphate (R-5P) "which comprises cultering a D-ribose phosphate-produc- "ing microorganism in an aqueous nutrient culture medium containing about 0.2 to 2.0% by weight (as phospho radical) of phosphate and about 0.01 to 0.2% by Weight (as magnesium ion) of magnesium salt, accumulating the aforesaid phosphates in the fermentation liquor and in the cells of the cultured microorganism, separating the fermentation liquid from the. remaining fermentation products and subjecting said liquid to fractional chromatography to separately recover said S-phospho-D-ribosyll-pyrophosphate, D-ribose-l,S-dyphosphate and D-ribose- '5-phosphate therefrom. The present process is not spetoany particular microorganisms or strains.

'The' present inventionis directed to a process for the recovery of D-ribose phosphates-namely, S-phospho-D- ribosyl-l-pyrophosphate (PRPP), D ribose 1,5 diphosphate (PRP) and D-ribose-S-phosphate (R-5-P)- from the fermentation liquor resulting from the cultivation of 'a microorganism in an appropriate culture medium and under suitable cluture conditions.

Ordinary processes hithertoin use for the production of D-ribose-S-phosphate from nucleic acid-related compounds depend upon decomposition, and hence are rather 'uneconomical. The production of R-5P by a fermentation method is known, but the possibility of producing ,PRPP and PRP through direct fermentation with the aid of microorganisms has not heretofore been reported. In fact, there is no known industrial process for the pro duction of PRPP and PRP. The present invention supplies such a process. Since this invention not only produces and makes possible the recovery of PRPP and PRP but also produces and makes possible the recovery of R-5-P by a microbiological fermentation and an associated recovery process, the entire process is rendered economically and industrially very feasible.

These three D-ribose phosphates are useful for biochemical reagents and nucleotide producing materials.

The present invention is based upon the observation that by subjecting the fermentation liquor, resulting from the culturing of a microorganism in an appropriate nutrient medium containing added magnesium salts and phosphrous salts (phosphates), to fractional chroma- 3,408,257 Patented 29, 1908 icetography, remark able yields" of all three D-ribose phosvphates-PRPF, PRP and R-S-P-are obtained. For reference, the chemical structures of PRPP, PRP andR-S-P are shown as follows:. v Q,

The production ratio of the said three D-ribose phosphates varies in accordance with the culture conditions, the culturing period and the species of microorganism. As to the latter, it is found that the present invention is not specific to any particular microorganism or strains, and that the invention is in fact applicable to strains of nn'broorganisms belonging to such various genera as Bacillus, Escherichia, Staphylococcus, Pseudomonas, Flavobacterium, Aerobacter, Leuconostos, Brevibacterium, Micrococcus, Corynebacterium, Arthrobacter, -Saccharomyces, Candida, Torula, Cryptococcus, Torulaspora, Mucor, Rhizopus, Penicillium, Paecilomyces, Gibberella, Trichoderma, Streptomyces, Micromonospora, and Eremothecium. Accordingly, the invention cannot be restricted to any particular microorganisms or to strains of any specific bacteriological classification. The invention is also applicable to nutrient-requiring mutants obtained by various mutation-inductive treatments.

The culture medium for use according to the present invention can be any medium which contains suitable amounts of carbon sources such as saccharides vor other carbon sources (for example, glucose, sucrose, starch, starch hydrolysates, molasses, gluconates, acetates, etc.), nitrogen sources (for example, such inorganic nitrogen sources as ammonium salts and nitrates, and such organic nitrogen sources as urea, amino acids, peptides and preparations containing these, including peptone, yeast extract, meats extract, Casamino acid, fish solubles and the like), and inorganic compounds (for example,

phosphates including potassium phosphate and ammoninesium salts, among the said inorganic compounds, are

added to the culture medium in a higher concentration than the usual concentration in fermentation media for general microorganisms; in other words, the phosphates and magnesium salts should be added in concentrations of 0.2 to 2.0%, and 0.01.to 0.2% (as magnesium ion), respectively, the said percentages being by weight when, de-

'gredients maybe addedin small amounts in the course of the fermentation.

Thefermentation is carried out ata culture temperature of 20 to 40? -C. under aerobic-conditions such as r 0.4 molarand finally 0.5 molarsodium formate buffer shaking culture and submerged culture under .aerobicagitation. Culture for 2 to 7 days leads to an accumulation of striking amounts of PRPP in the culture liquor and in the cells of the culturedmicroorganisms, PRP and RP b'eing accumulated at the same time.

After the culture has been completed, the PRPP, PR aridR-S-Pcanbe individually recovered from the culture liquor and the said cells by fractional chromatogra- -'phyas illustrated in the-following examples. Use is made in this regard of a column of an appropriate strongly basic anion exchange resin (formate-type, acetate-type or cl-type), such eg. as Dowex-l, Amberlite-IRA, Duolite-A Or-DiaiOn-SA. Elution -is thereafter effected with eluants which separately elutethe objective substances. Suitable eluants comprise, for example, formic acid-sodiurn or ammonium formate, ac'e'tic acid-sodium 'or ammonium acetate, or hydrochloric acid-sodium or ammoni- EXAMPLE 1 Brevibrzcterirtm ammoniagenes KY3454 (ATCC 6872) as the s eed was incubated in 300 milliliters of an aqueous culture medium containing 2% of glucose, 1% of peptone, 1% of meat extract, 0.25% of NaCl, and 0.2% urea in a 2-liter conical flask, which was sterilized, and maintained at a pH valve of 7.5 at 28 C. for 24 hours.

The resulting culture was inoculated into the fermentation medium in the ratio of 10% by volume relativeto the latter. Forty milliliters of the fermentation medium was placed in a 500 milliliter Sakaguchis flask and used after sterilization.

Using the fermentation medium having the composition described below, shaking culture was conducted at 28 C.

The fermentation medium was as follows:

Glucose pct 12 Urea pct 0.6 KH PO; v pct 1.2 K2HPQ4 pct ii-Alanine -'y/ml 5 MgSO -7H O pct 0.5 Biotin y/literm. 30 CaCl -2H O pct 0.01 Meat extract pct 02 FeSO -7H O pct 0.001

In this way, in the fermentation liquor cultured for 72 hours, R-5-P, PRPand PRPP were accumulated in a yield of 15.4 mgJml. calculated as R-5-P. In addition, the accumulation of these phosphates was observed also in the cells of the cultured microorganisms.

To 100 milliliters of the filtrate obtained by removing the said cells from the former fermentation liquor, calcium hydroxide was added and the deposited inorganic phosphorus compounds were removed by centrifugation. The resulting supernatant liquid was passed through a columnof Dowex-l (formic acid type), the ribose phosphates being absorbed and then eluted with 0.2 molar-,

solutions (pH 5.0) in order.

R-S-P, and both of PRP and PRPP were contained in the 0.2 molar-sodium formate eflluent and in the 0.5 molarsodium formateetfiuent, respectively. Each fraction was passed through a columniof-Diaionis KlNo'. v1 (H type), neutralized topH' 7.0, -and then concentrated under reduced pressure. a

The concentrate was treated with asmallfamol t of carbon powder and the pH ther of was adjusted to 8.3 with sodium hydroxide. Tothe c'onc ritrate'treated thus, barium acetate (20%) in anamount 1.2 times the calculated amount and alcohol (four-fold)" we re added to deposit the corresponding salts. The precipitates were dried under diminished pressure to afford R- and PRPP in the yields of 311 milligrams, 2S0m'1ll1gr arns, and29 'grams','respe'ctively, calculated as' the indivldu al anhydrous barium salts;

EXAMPLE 2 Using Micrococcus glutamicus ATCC l4995 the seed, the seed cultivation was carried out in thesa'meway as described precedingly and theresulting culturejwas inoculated into the fermentation medium in the-ratioof 10% by volume relative to the latteh'A fermentation medium having the following composition was employed:

F6804 -pct- .pH: Adjusted to .7.3 before sterilization.

The fermentation was carriedout by use of 3v liters of the cultured medium, which was sterilized in a 5-liter. jar fermentor, by submerged culture under aeration-agitation. During the course of the-culture, the pH was adjusted to 7.0. When. the incubation was conducted at 34. C. for 48 hours, PRPP, PRP and R-S-P were accumulated. .in

the fermentation liquor in a yield of 10.2 mg./ml'., calculated as R-S-P. In addition, such accumulationwas found also in the cells of the cultivated microorganism. From 2 liters of the fermentation liquor, R-S-P, PRP and PRPP were fractionated by the same procedure-as in Example 1 and separated in yields of 1.1 grams," 4.2 grams, and 41.2 grams, respectively, calculated as the individual anhydrous barium salts. i u

EXAMPLE 3 When by using the following fermentation medium, variouskinds of microorganisms were cultured under the same conditions as in Exampled for 48'hours, 96 hours, and 6 days in the cases of bacterium, .yeast and actinomyces, and mold, respectively, PRPP, PRP,- and rises culturing the said microorganism in a nutrient medium which comp phate-producing microorganism TABLE 1 strain used therefore containing 0.2 to 2.0% (as phospho radical) of PRPP PR1 R-5- phosphate and 0.01 to 0.2 (as magnesium ion) of magfndyn- .g-den-nn w 1 C S S t 33mm mmmm mmnm z m ,wmrm. Puma m w mam 5 a c 3 0 Emm mmmmm o w s e u e m r. hi a .n m a m md m Wm bwm w a mM mfi a own magma? .d .w m mn q Dwm p a s wm m B m m H mm w om d m m m mm a N l t O u mm m am o mm on m n m .1. Ofi OP 0 a e t P n d m mm mm n wimma wa we m m a man 6 h a a m w r n m. a b m m e m m m m s .u 0 0 S n (r S O.H m h V 0 0 w E 1% 0 0 o r 0 t o cT 3.1 h PD u 6C5 1 O I g H .1 n e y A H A a m .1 P 8 P S .1 h 1 P e m 8 H e r. m mmwmmy w mw .mwK V. m c am MU, m Pm m md w m D 7 b H M .1 V ..I g a S 0 C mfi f 8 1 E 6 6 8 e a 6 r :H O h r Ora COS n (s a LY T9 mam wmm mw awmmw mm m mm e o 01 m 0 a t t c o m U 2 m H m 0 a 8 Wk n A l e m m ,e n 2 v o a t P e s 1 a r 0 a e r a Y o0 ofi a d 8 0 fim m m m lmmflwwm m k 1 mhm mph az mnc hs a u u m 1 .3 1 5 .1 i a w a m o mm wm 0a2ac mvr l fl 3, a q hh h r Y m 3 ncmhwmPP mmomkpw y .wadb .U 5 0 5 m H 2 2 3 s a r P .w m8 r 5 An m E R E CLQ N H m E a 2 F m M u 2 R m d m m w w H T 6 N 0 an m mm T Uk 1 r. E o W m w m N A What is claimed is:

1. A process for the concomitant production and individual recovery of 5-phospho-Dribosyl-l-pyrophosphate, D-ribose-1,5 diphosphate and D-ribose-S-phosphate directly by the fermentatlon of a D-r1bose phos- U.S. DEPARTMENT OF COMMERCE PATENT OFFICE Washington, 0.6. 20231 UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3,408,257 October 29, 1968 Yoshiaki Kawamori et a1.

It is certified that error appears in the above identified patent and that said Letters Patent are hereby corrected as shown below:

In the heading to the printed specification, line 8, "Koyo" should read Kogyo Signed and sealed this 17th day of March 1970.

(SEAL) Attest:

WILLIAM E. SCHUYLER, JR.

Commissioner of Patents Edward M. Fletcher, Jr.

Attesting Officer 

